Novel Anti-carbohydrate Antibodies Reveal the Cooperative Function of Sulfated N- and O-Glycans in Lymphocyte Homing

• Published in: The Journal of biological chemistry 2010-12, Vol.285 (52), p.40864-40878
• Main Authors: Hirakawa, Jotaro, Tsuboi, Koichiro, Sato, Kaori, Kobayashi, Motohiro, Watanabe, Sota, Takakura, Atsushi, Imai, Yasuyuki, Ito, Yuki, Fukuda, Minoru, Kawashima, Hiroto
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Monoclonal - immunology; Antibodies, Monoclonal - pharmacology; Carbohydrate; Carbohydrate Function; Carbohydrate-binding Protein; Cell Adhesion - drug effects; Cell Adhesion - immunology; CHO Cells; Cricetinae; Cricetulus; Glycobiology and Extracellular Matrices; Glycoprotein; Glycoproteins - genetics; Glycoproteins - immunology; Glycoproteins - metabolism; Glycosylation; Humans; Immunologic Surveillance - drug effects; Immunologic Surveillance - immunology; l-Selectin; Lewis Blood Group Antigens; Lymphocyte Homing; Lymphocytes - enzymology; Lymphocytes - immunology; Mice; Mice, Knockout; Oligosaccharides - biosynthesis; Oligosaccharides - genetics; Oligosaccharides - immunology; Sulfotransferase; Sulfotransferases - biosynthesis; Sulfotransferases - genetics; Sulfotransferases - immunology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110.167296

Cell surface glycans play pivotal roles in immune cell trafficking and immunity. Here we present an efficient method for generating anti-carbohydrate monoclonal antibodies (mAbs) using gene-targeted mice and describe critical glycans in lymphocyte homing. We immunized sulfotransferase GlcNAc6ST-1 and GlcNAc6ST-2 doubly deficient mice with sulfotransferase-overexpressing Chinese hamster ovary cells and generated two mAbs, termed S1 and S2. Both S1 and S2 bound high endothelial venules (HEVs) in the lymphoid organs of humans and wild-type mice, but not in those of doubly deficient mice. Glycan array analysis indicated that both S1 and S2 specifically bound 6-sulfo sialyl Lewis X and its defucosylated structure. Interestingly, S2 inhibited lymphocyte homing to peripheral lymph nodes by 95%, whereas S1 inhibited it by only 25%. S2 also significantly inhibited contact hypersensitivity responses and l-selectin-dependent leukocyte adhesion to HEVs. Immunohistochemical and Western blot analyses indicated that S1 preferentially bound sulfated O-glycans, whereas S2 bound both sulfated N- and O-glycans in HEVs. Furthermore, S2 strongly inhibited the N-glycan-dependent residual lymphocyte homing in mutant mice lacking sulfated O-glycans, indicating the importance of both sulfated N- and O-glycans in lymphocyte homing. Thus, the two mAbs generated by a novel method revealed the cooperative function of sulfated N- and O-glycans in lymphocyte homing and immune surveillance.